Involvement of basic amino acid residues in transmembrane regions 6 and 7 in agonist and antagonist recognition of the human platelet P2Y 12-receptor

Abstract

The P2Y 12-receptor plays a prominent role in ADP-induced platelet aggregation. In the present study, we searched for amino acid residues involved in ligand recognition of the human P2Y 12-receptor. Wild-type or mutated receptors were expressed in 1321N1 astrocytoma cells and Chinese hamster ovary (CHO) cells. There were no major differences in cellular expression of the constructs. Cellular cAMP production and cAMP response element (CRE)-dependent luciferase expression was increased by isoproterenol (astrocytoma cells) or forskolin (CHO cells). In cells expressing wild-type receptors, R256K or S101A mutant constructs, 2-methylthio-ADP inhibited the induced cAMP production with IC 50 concentrations of about 0.3 nM. In cells expressing R256A constructs, the IC 50 concentration amounted to 25 nM. In cells expressing H253A/R256A, Y259D and K280A constructs, 2-methylthio-ADP failed to affect the cellular cAMP production. Moreover, in cells expressing Y259D and K280A constructs, 2-methylthio-ADP did also not change the forskolin-induced CRE-dependent luciferase expression and caused only small increases in the serum response element-dependent luciferase expression. The antagonist cangrelor had similar potencies at wild-type receptors and R256A constructs (apparent p K B-value at wild-type receptors: 9.2). In contrast, reactive blue-2 had a lower potency at the R256A construct (apparent p K B-value at wild-type receptors: 7.6). In summary, the data indicate the involvement of Arg256, Tyr259 and, possibly, H253 (transmembrane region TM6) as well as Lys280 (TM7) in the function of the human P2Y 12-receptor. Arg256 appears to play a role in the recognition of nucleotide agonists and the non-nucleotide antagonist reactive blue-2, but no role in the recognition of the nucleotide antagonist cangrelor.